Method of preventing T cell-mediated responses by the use of the major histocompatibility complex class II analog protein (map protein) from Staphylococcus aureus

ABSTRACT

A method of immunomodulating the T cell response in Staphylococcal bacteria is provided wherein an effective amount of the Map protein from  Staphylococcus aureus  is administered to a host to prevent or suppress the T cell response. The present method may be utilized with either the Map protein or an effective subdomain or fragment thereof such as the Map 10 or Map 19 protein. The present invention is advantageous in that suppression or prevention of the T cell response in a host can prevent or ameliorate a wide variety of the pathogenic conditions such as T cell lymphoproliferative disease and toxic shock syndrome wherein the overstimulation of T cells needs to be suppressed or modulated.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisionalapplication Serial No. 60/260,523, filed Jan. 10, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates in general to the utilization ofmajor histocompatibility complex class II analog protein, or “Map”protein, and its biologically effective fragments and domains thereof,in therapeutic methods to combat conditions associated with T cellproliferation, and in particular to the use of the Map protein andeffective or active fragments thereof, including the Map10 or Map19protein, in methods of suppressing or modulating T cell-mediatedresponses where necessary to alleviate a pathogenic condition.

BACKGROUND OF THE INVENTION

[0003]Staphylococcus aureus (SA) is an opportunistic pathogen that cancause a wide spectrum of infections from superficial local skininfections to life-threatening systemic infections that can affectinternal organs and tissues. In addition, bacterial arthritis, as wellas acute and chronic osteomyelitis caused by haematogenous spread or bydirect inoculation in open trauma or surgical intervention such asinternal fixation or joint replacement, affect hundreds of thousands ofpatients each year (1-6). SA is also a major cause of infectionsassociated with indwelling medical devices, such as catheters andprosthesis (6). The cost to society in patient care, which ofteninvolves extended hospital stays and repeated surgery, can be estimatedat several billion dollars per year. With the documented emergence ofmultidrug resistance SA strains, the threat of this widely distributedpathogen is now appreciated and novel therapies for treatment andprevention are needed.

[0004] The successful colonization of the host is a process required formost microorganisms, including S. aureus, to cause infections in animalsand humans. Microbial adhesion is the first crucial step in a series ofevents that can eventually lead to disease. Pathogenic microorganismscolonize the host by attaching to host tissues or serum conditionedimplanted biomaterials, such as catheters, artificial joints, andvascular grafts, through specific adhesins present on the surface of thebacteria. MSCRAMM™S (Microbial Surface Components Recognizing AdhesiveMatrix Molecules) are a family of cell surface adhesins that recognizeand specifically bind to distinct components in the host's extracellularmatrix. Once the bacteria have successfully adhered and colonized hosttissues, their physiology is dramatically altered and damagingcomponents such as toxins and proteolytic enzymes are secreted.Moreover, adherent bacteria often produce a biofilm and quickly becomemore resistant to the killing effect of most antibiotics.

[0005]S. aureus is thus known to express a repertoire of differentMSCRAMM™S that can act individually or in concert to facilitatemicrobial adhesion to specific host tissue components. A search for suchMCSRAMM's which recognized host components uncovered a 72-kDa proteinidentified as the major histocompatibility complex class II analogprotein, or “Map” protein, a surface localized protein expressed byvirtually every S. aureus strain (7). Cloning and sequencing of the geneencoding the Map protein revealed a protein consisting of roughly110-amino acid-long domains repeated six times with each domaincontaining a 31 amino acid-long subdomain with homology to MHC Class II.If conservative amino acid substitutions were included, the respectivesubdomains were 61, 65, 52, 59, 52 and 45% similar to the amino-terminalend of the b chain of many MHC class II proteins from differentmammalian species (8).

[0006] However, previous studies varied with regard to how the Mapprotein affected immune function, and thus it would be highly desirableto utilize the Map protein so as to affect the T cell immune responsesin cases where pathogenic conditions result from a proliferation of Tcells.

SUMMARY OF THE INVENTION

[0007] Accordingly, it is an object of the present invention to providea method of utilizing the S. aureus Map protein, or effective fragmentsand domains thereof, to suppress or modulate the T cell response inhuman or animal patients.

[0008] It is also an object of the present invention to provide andutilize binding subdomains of the S. aureus MAP protein, including theMap19 protein, in methods of treating or protecting against conditionsassociated with the overstimulation of T cells.

[0009] It is also an object of the present invention to provide isolatedMap proteins and active fragments and regions therefrom to prevent Tcell-mediated responses in human or animal patients thus reduce orprevent pathogenic and deleterious conditions that arise because of Tcell proliferation.

[0010] These and other objects are provided by virtue of the presentinvention which provides methods of utilizing the Map protein and/or itsbinding subdomains or other effective fragments thereof, to suppress ormodulate the T cell immune response in human or animal patients undercircumstances where such a response has deleterious consequences. Useand administration of an effective amount of the Map protein or itseffective subdomains or fragments thereof can thus be utilized in a hostto reduce T cell proliferation and achieve a significant reduction in Tcell-mediated processes such as delayed-type hypersensitivity (DTH).Suitable compositions and vaccines based on the isolated MAP protein andits effective regions and subdomains, such as the Map10 and Map19proteins, as well as methods for their use, are also contemplated by thepresent invention.

[0011] These embodiments and other alternatives and modifications withinthe spirit and scope of the disclosed invention will become readilyapparent to those skilled in the art from reading the presentspecification and/or the references cited herein, all of which areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0012]FIGS. 1a-1 c are graphic representations showing the Map-inducedinhibition of DTH in accordance with the present invention.DpbA-immunized mice were treated with either native Map (A) orrecombinant Map19 (B-C) on the day of immunization (day 0) and on days2, 4, and 6 post immunization. On day 7, BALB/c (A-B) and C3H/Hen (C)mice were challenged with DbpA and footpads were measured 0 and 24 hafter challenge. Mice treated with supernatant from Map⁺ V SA (A) orrecombinant Map19 (B-C) had a significantly reduced DTH responsecompared to immunized and challenged mice (p<0.0001*; Student's t test).Data are expressed as the mean±SE of 5 mice.

[0013]FIG. 2 shows the Map19 dose-response for inhibition of DTH inaccordance with the present invention. DbpA-immunized mice were treatedwith various doses of Map19 (25-200 μg) or ACE19 (200 μg) as describedpreviously. On day 7, mice were challenged with DbpA and footpads weremeasured 0 and 24 h after challenge. Significant values are indicated byan * (Students t test). Data are expressed as the mean±SE of 5 mice.

[0014]FIG. 3 is a graphic representation of tests showing thatadoptively transferred T cells from Map-treated mice do not elicit a DTHresponse in naive mice. DbpA-immunized mice were treated with eitherMap19 or SdrF as described above. On day 7, mice were sacrificed andspleens were harvested and enriched for T cells by nylon woolpurification. 5×10⁷ cells were injected i.p. into syngeneic recipients.24 h later, recipient mice were challenged with DbpA and the DTHresponse was assessed as described above. DbpA-immunized andDbpA-immunized, SdrF-treated mice developed a significant DTH responsecompared to unimmunized but challenged mice (p<0.04*; Student's t test).DbpA-immunized, Map19-treated mice had a significantly reduced DTHresponse compared to the other treatment groups (p<0.001**; Student's ttest). Data are expressed as the mean±SE of 5 mice.

[0015]FIG. 4 is a graphic representation of the Map-induced inhibitionof T cell proliferation using the method of the present invention. Inthis test, BAT2.2 T cell proliferation was measured after 40 h inculture in the presence of APCs and antigen in the presence of variousproteins. 100 μg of each protein was added per well. Data are expressedas the mean absorbance±SE of triplicate wells.

[0016]FIG. 5 shows Map-induced apoptosis of BAT2.2 T cells in accordancewith the present invention. BAT2.2 cells (5 U IL-2/ml) were incubated inmedia alone (lane 1), or in the presence of either 100 μg Map19 (lane 2)or ACE40 (lane 3). DNA from U937 cells were used as a positive control(lane 5).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In accordance with the present invention, there are providedmethods and immunogenic compositions for suppressing, preventing orimmunomodulating T cell-mediated responses in human or animal patients.In the preferred methods of the present invention, an effective amountof an isolated natural or recombinant Map protein or an active fragmentor domain therefrom such as the Map10 or Map19 protein, is utilized inan amount effective to achieve such suppression or modulation. The MAPprotein is a surface localized protein expressed by virtually every S.aureus strain. McGavin et al (7) originally identified the 72 kDasurface protein, from S. aureus strain FDA 574, that binds a variety ofhost proteins including BSP, fibrinogen, fibronectin, vitronectin, andthrombospondin. The gene, designated map, was cloned and sequenced (U.S.Pat. No. 5,648,240, incorporated herein by reference).

[0018] Reinfection of humans with SA is one of the hallmarks of diseasescaused by this pathogen and the roles of acquired and innate immunity inprotection against infection vary with the many manifestations ofdisease resulting from SA infections (25-28). While SA infectionsaffecting the skin appear to be exacerbated by strong cellularresponses, it is clear that cellular immunity is critical inorchestrating the clearance of systemic SA infections and in preventingreinfection with the same or similar pathogens (29-33). One possiblereason for recurring SA infections is the reduction in chemotactic,phagocytic and bactericidal functions of polymorphonuclear leukocytesfrom patients with chronic or recurrent SA infections (27, 30, 33).Whether this is a function of the bacterial infection or a preexistingcondition in these individuals is not known (27, 30, 33).

[0019] Regardless, the presence of SA-immunoregulatory moleculessuggests that these bacteria have the potential to counteract or evadehost defense mechanisms. Both superantigens and protein A produced by SAduring an infection serve immune-evasion functions. Superantigens canactivate between about 5-20% of T cells by directly binding to both themajor histocompatibility complex (MHC) class II molecules onantigen-presenting cells and to the T cell receptor (TCR) on T cells.This interaction can initiate apoptosis in T cells and thymocytes invivo and in vitro. The in vivo effects of such massive T cellstimulation often results in disease (e.g., toxic shock syndrome andfood poisoning in humans) (34). Protein A, while less harmful to thehost compared to superantigens, may also serve as a means of immuneevasion by binding to the Fc fragment of immunoglobulins (i.e. IgG)resulting in loss of antibody function.

[0020] As described herein, the present inventors have shown that theMap protein and its effective regions or subdomains such as Map10 andMap19 appear to function as immune modulators with the capacity toaffect host immune responses such as during SA infections. In accordancewith the present invention, compositions containing the Map protein asdescribed further below have the capacity to interfere with T cellactivation and/or proliferation and can serve to potentiate survival inmammals of varied genetic backgrounds.

[0021] Studies in accordance with the present invention have shown thatMap serves as an immunomodulatory protein as evidenced in doubleinfection studies in which a primary infection with Map³¹ SA conferredsignificant protection against reinfection with Map⁺SA. This contrastssignificantly with SA-induced pathology from mice receiving primary andsecondary infections with Map⁺. Accordingly, T cell-mediated responsesin Map⁺-infected mice appear to be abrogated by the presence of Mapcompared to Map⁻SA-infected mice which develop cell-mediated immunityover the course of infection. Map⁻SA infection, which is cleared overtime, results in a memory response capable of controlling a secondaryMap⁺infection. That a primary Map⁻SA infection conferred significant butnot complete protection against Map⁺SA challenge suggested that thedelicate balance between an anamnestic response and Map-mediatedimmunomodulation could be affected by the challenge dose. Inhibition ofDTH responses directly or as a result of adoptively transferred T cellsfrom Map-treated mice combined with the in vitro effects of Map on Tcell proliferation, have evidenced a direct involvement of Map with Tcells resulting in apoptosis. Flow cytometric analysis of fluoresceinisothiocyanate (FITC)-labeled Map19 revealed binding to 100% of BAT2.2 Tcells.

[0022] Additional tests of nylon wool-purified naive T cells cultured inthe presence of Map19 were not induced to either proliferate or undergoapoptosis. Furthermore, proliferation of naive T cells as a result ofincubation with concanavalin A or by antibody-cross-linking of the TCRwas not inhibited by Map. This result evidenced that activated T cellsbut not naive T cells are susceptible to Map and that T cellproliferation via ‘non classical’ pathways bypasses the Map-mediatedinhibition of T cell proliferation. The present data provides evidencethat Map functions as an immunoregulatory protein during SA infectionsand it appears that this protein is yet another weapon used by SA toescape immune recognition and clearance.

[0023] Accordingly, in accordance with the present invention, methods ofutilizing an effective amount of the Map protein or its active regionsor subdomains such as Map10 or Map19 are provided which can be used totreat or prevent T cell-mediated responses.

[0024] In addition, the administration of suppressive orimmunomodulatory effective amounts of an isolated and/or purified S.aureus Map protein or one of its effective regions such as Map10 orMap19 can be utilized in methods of treating or preventing pathologicalconditions associated with overstimulation of T cells such as toxicshock syndrome. In accordance with the present invention, a method isprovided which comprises administering to a human or animal patient inneed of such treatment an effective amount of an isolated natural orrecombinant Map protein. By Map protein is meant the whole natural orrecombinant Map protein, or any effective or otherwise immunologicallyactive fragment, fraction, domain, subdomain or region thereof whichalso has effective immunogenic properties so as to prevent or suppress aT cell-mediated response in the patient. In accordance with theinvention, one such region is the Map19 protein, the nucleic acidsequence of which is provided herein as SEQ ID NO: 1, and the amino acidsequence is provided as SEQ ID NO: 2. Another such region is the Map10protein, the nucleic acid sequence of which is provided herein as SEQ IDNO: 3, and the amino acid sequence is provided as SEQ ID NO: 4.Accordingly, the present invention also relates to methods ofadministering immunologically effective amounts of an isolated and/orpurified S. aureus Map10 or Map19 protein so as to be utilized inmethods of treating or preventing pathological conditions associatedwith T cell proliferation or other T cell-mediated responses.

[0025] As would be recognized by one of ordinary skill in the art,compositions containing an effective amount of the Map protein, theMap10 protein or the Map19 protein can be prepared and administered to ahuman or animal patient in need of such treatment, particularly thosepatients requiring treatment or prevention of pathological conditionsand other diseases associated with the T cell-mediated response.

[0026] By effective amount, it is recognized that the preferred dose foradministration of a composition containing the Map, Map10 or Map19protein in accordance with the present invention is that amount will beeffective in preventing or modulating the T cell response, and one wouldreadily recognize that this amount will vary greatly depending on thenature of the infection and the condition of a patient. Accordingly, an“effective amount” of the Map protein to be used in accordance with theinvention is intended to mean a nontoxic but sufficient amount of theagent, such that the desired prophylactic, immunological or therapeuticeffect is produced.

[0027] As one of skill in the art would recognize, the exact amount ofan effective composition that is required will thus vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the condition being treated, the particularcarrier or adjuvant being used and its mode of administration, and thelike. Accordingly, the “effective amount” of any particular compositionwill vary based on the particular circumstances, and an appropriateeffective amount may be determined in each case of application by one ofordinary skill in the art using only routine experimentation. Forexample, it is contemplated that an effective amount may be as little asabout 15 μg in an application in order to achieve suppression of the Tcell response, but this amount may be increased in cases wherein ahigher dosage regimen is required. The dose should thus be adjusted tosuit the individual to whom the composition is administered and willvary with age, weight and metabolism of the individual. The compositionsmay additionally contain stabilizers or pharmaceutically acceptablepreservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercurysodium salt) (Sigma Chemical Company, St. Louis, Mo.).

[0028] The compositions of the invention may also be used as vaccineswhich will be useful in generating antibodies in a host patient whichalso may be useful to treat or preventing conditions associated withstaphylococcal infection or T cell proliferation. As would be recognizedby one skilled in this art, a vaccine may be prepared for administrationin a number of suitable ways, such as by parenteral (i.e.,intramuscular, intradermal or subcutaneous) administration ornasopharyngeal (i.e., intranasal) administration, and the generalcomposition comprises the effective isolated Map, Map10 or Map19 proteinalong with a pharmaceutically acceptable vehicle, carrier or excipient.In one such mode, the vaccine is injected intramuscularly, e.g., intothe deltoid muscle, however, the particular mode of administration willdepend on the nature of the bacterial infection and the condition of thepatient. In addition, antibodies from the Map protein may be obtainedand isolated in conventional ways by the introduction of the appropriateMap protein in an appropriate host. In any event, the vaccines of theinvention may be combined with any of a variety of pharmaceuticallyacceptable vehicles, carriers or excipients, such as water or a bufferedsaline, that are well known to those of ordinary skill in the art. Inaddition, the vaccine may be lyophilized for resuspension at the time ofadministration or in solution.

[0029] In carrying out the method of the present invention, theisolation and/or purification of the Map protein, Map10 or of the Map19protein, or other active fragments or domains of the Map protein, can beaccomplished in a number of suitable ways as would be recognized by oneskilled in the art. For example, the Map protein and its effectivesubregions, such as Map10 or Map19, may be obtained and/or purifiedrecombinantly using conventional techniques well known in the industry.With regard to the Map19 protein (SEQ ID NO: 2), one such suitablemethod would be through expression in E. coli (e.g., JM101 from Qiagen,Chatsworth, Calif.) harboring the appropriate plasmid (11-16). In thismethod, E. coli was grown at 37° C. in LB containing the appropriateantibiotics until they reached an A₆₀₀ of 0.6 (17).Isopropyl-β-D-thiogalactopyranoside (IPTG) (Life Technologies) was addedto a final concentration of 0.2 mM, and the cells were incubated at 37°C. for an additional 4 hours. Cells from a 1 L culture were harvested bycentrifugation and resuspended in 10 ml “binding buffer” (BB) (20 mMTris HCl, 0.5 M NaCl, 15 mM imidazole, pH 8.0) and lysed in a Frenchpressure cell at 11,000 pounds/inch² (13). The lysate was centrifuged at40,000×g for 15 min and the supernatant filtered through a 0.45 mfilter. A 1 ml iminodiacetic acid Sepharose column (Sigma, St. Louis,Mo.) was charged with 75 mM NiCl₂.6H₂O and equilibrated with BB. Thefiltered supernatant was applied to the column and washed with 10volumes of BB, then 10 volumes of BB containing 60 mM imidazole. Thebound proteins were eluted with BB containing 200 mM imidazole, dialyzedagainst PBS containing 10 mM EDTA, then dialyzed against PBS (13).Protein concentrations were determined by the Bicinchoninic Acid (BCA)Protein Assay (Pierce) and proteins were stored at −20° C. until use.

[0030] In addition to obtaining isolates of the Map protein throughrecombinant means, natural isolates of the Map protein may be obtainedfor use in the present invention by a number of suitable means as well.For example, the natural Map protein can be extracted using standardmethods. In one such suitable method, Map⁺SA and Map⁻SA were grownovernight as described above. Bacteria were pelleted by centrifugationand resuspended in 1 M LiCl (one tenth of the original media volume).The suspension was incubated at 42° C. with shaking for two hours. Thebacteria were pelleted and the supernatant was removed and quantifiedfor protein by UV spectrophotometry using 1 M LiCl as a blank. Extractedproteins were diluted to 0.2-mg/ml in PBS and passed through a0.45-micron filter for sterilization prior to i.p. injection (7).

[0031] As indicated above, and in the examples below, the method of thepresent invention is carried out by administering effective amounts tohuman or animal patients so as to achieve the desired prophylactic,immunological or therapeutic effect via the suppression, reduction ormodulation of the T cell response, and such effective amounts would bedetermined through routine means as indicated above for a particularpatient based on factors such as type and size of patient, type ofinfection, level of virulence, etc. For example, it is contemplated thatformulations with as little as 15 μg of an isolated Map protein, orMap10 or Map19, may be effective in achieving the suppression ormodulation of the T cell response.

[0032] Map's ability to impede the development of cell-mediated immunitythus evidences that recombinant Map or formulations thereof as describedabove may have tremendous potential therapeutic value in a wide varietyof clinical and pathologic conditions. For example, certain T celllymphoproliferative diseases may be potentially treated with Map; theseinclude thymoma, T lymphoblastic lymphoma, T chronic and acutelymphoblastic leukemia (20-30%), mycosis fungoides (Sezary's syndrome),T cell type of hairy cell leukemia, HTLV-associated Japanese, Caribbeanand American adult T cell leukemia/lymphoma, and approximately 30% ofnon-Hodgkin's lymphomas (NHL). Non-Hodgkin's lymphoma is the fifth mostfrequent malignancy in the United States with more that 55,000 casesdiagnosed in 1997. The incidence of this disease has increased 3-5% overthe last two decades. A variety of treatments aimed at reducing cellproliferation and suppressing immune function in cases oflymphoproliferative diseases, in particular NHL, can include purineanalogues, chemotherapy, surgery, glucocorticoids, α-2-recombinantinterferon, and recombinant interferon γ(16-19).

[0033] In addition to neoplastic lymphoproliferative disorders,autoimmune lymphoproliferative syndrome associated with defects of theFas gene result in uncontrolled activation of lymphocytes which lead tolymphadenopathy and progression of autoimmune disease. In murine modelsof autoimmunity, treatment with antibodies against T cells will retarddisease progression as long as the treatment is continued, however,these treatments are not available to humans (20). In addition,long-term administration of agents such as anti-inflammatory agents,immunosuppressants, and cytotoxic agents that have previously been usedto treat autoimmune disease such as systemic lupus erythromatosis andreactive arthritis can result in a plethora of side-effects.

[0034] Therapeutic applications for Map may also be available in variousconditions resulting from microbial infections. While specific immunityto extracellular bacteria is primarily humoral in nature, T cellresponses to extracellular microbes consist of CD4⁺ T cells respondingto antigens associated with MHC II molecules. Potential injuriousconsequences resulting from this type of infection are a result ofbacterial toxins (super antigens) that can stimulate large numbers ofCD4⁺ T cells. These proliferating T cells can produce large quantitiesof cytokines that result in abnormalities that are similar to septicshock.

[0035] Patients who survive the critical phases of Listeriamonocytogenes infections develop activated T lymphocytes that promotethe formation of granulomas. Although both CD4⁺ and CD8⁺ T cells areactivated during Listeriosis, the protective efficacy of CD4⁺ cells isminor compared to that of CD8⁺ cells. CD4⁺ cells, however, are necessaryfor granuloma formation, DTH, and splenomegaly and their presence isclosely associated with the production of cytokines in Listeria-inducedantigen-specific inflammatory phenomena (21-23).

[0036] Specific immune responses to parasites (protozoa, helminthes, andectoparasites) are usually CD4⁺-mediated. In some cases, immuneresponses to parasites can also contribute to tissue injury. Someparasites and their byproducts can cause granuloma formation withconcomitant fibrosis. The helminth Schistosoma mansoni release eggs intothe blood stream, many of which remain lodged in the liver. The hostimmune response to the eggs is CD4⁺-mediated and results in a DTHresponses against the eggs followed by granuloma formation.Granuloma-associated fibrosis resulting from this immune response leadsto disruption of venous blood flow in the liver, portal hypertension,and cirrhosis.

[0037] Alternate treatment modalities for modulating T cell responsesgone awry is of significant concern since some treatments e.g.immunosuppressants and chemotherapy have detrimental side effects andsome conditions resulting from bacterial infections (e.g. septic shock)do not have established treatment protocols.

[0038] Even further, the Map compositions in accordance with the presentinvention may be useful in treatment of T cell proliferative conditionssuch as poison ivy. In the preferred mode, the effective amount of theMap protein, or active regions such as Map10 or Map19, would be used ina cream or other dermatologically acceptable form and applied on theaffected area.

[0039] In short, the present invention can thus be utilizedadvantageously as a means of treating or preventing pathologicalconditions associated with the T cell immune response and will be usefulin suppressing or modulating the T cell-mediated responses in a human oranimal patient so as to treat, prevent or ameliorate a wide variety ofconditions caused by T cell-mediated responses.

EXAMPLES

[0040] The following examples are provided which exemplify aspects ofthe preferred embodiments of the present invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples which follow represent techniques discovered by theinventors to function well in the practice of the invention, and thuscan be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1

[0041] Overview

[0042]Staphylococcus aureus (SA) expresses a 72-kDa protein with thecapacity to bind to a variety of extracellular matrix components (ECM),suggesting that at least one role for this protein involves adherenceand colonization of host tissues. Analysis of Map, however, alsorevealed homologies to a segment of the peptide-binding groove of the bchain of the major histocompatability class (MHC) II mammalian proteins.Map-deficient SA (Map⁻ SA) were generated to examine Map's role in theinfection process. Map⁻SA-infected mice presented with significantlyreduced levels of arthritis, osteomylitis, and abscess formationcompared to Map⁺SA-infected control animals. Furthermore,Map⁻SA-infected mice challenged with Map⁺SA were significantly protectedagainst SA-induced pathology compared to mice infected and challengedwith Map⁺. Native and recombinant forms of Map were tested for theirability to interfere with T cell response in vivo and in vitro. T cellsor mice treated with recombinant Map had reduced levels of T cellproliferation and significant reduction of the delayed-typehypersensitivity (DTH) response to challenge antigen, respectively. Thedata presented here evidence a role for Map as an immunomodulatoryprotein which may play a role in persistent SA infections and thus mayfunction to potentiate SA survival in mammals by affecting the host'scellular immune responses.

[0043] Background

[0044]Staphylococcus aureus (SA) is an opportunistic pathogen that cancause a wide spectrum of infections from superficial local skininfections to life-threatening systemic infections that can affectinternal organs and tissues. In addition, bacterial arthritis, as wellas acute and chronic osteomyelitis caused by haematogenous spread or bydirect inoculation in open trauma or surgical intervention such asinternal fixation or joint replacement, affect hundreds of thousands ofpatients each year (1-6). SA is also a major cause of infectionsassociated with indwelling medical devices, such as catheters andprosthesis (6). The cost to society in patient care, which ofteninvolves extended hospital stays and repeated surgery, can be estimatedat several billion dollars per year. With the documented emergence ofmultidrug resistance SA strains, the threat of this widely distributedpathogen is now appreciated and novel therapies for treatment andprevention are needed.

[0045] A search for SA adhesins recognizing host components uncovered a72-kDa protein capable of binding a variety of host proteins (7).Cloning and sequencing of this gene revealed a protein consisting of110-amino acid-long domains repeated six times with each domaincontaining a 31 amino acid-long subdomain with homology to MHC class II.If conservative amino acid substitutions were included, the respectivesubdomains were 61, 65, 52, 59, 52, and 45% similar to theamino-terminal end of the chain of many MHC class II proteins fromdifferent mammalian species (8).

[0046] The present work supports a role for Map as an immunomodulatoryprotein. Mice infected with SA genetically manipulated to be deficientin Map (Map⁻SA) have significantly reduced levels of arthritis andabscess formation (heart and kidneys) following reinfection withwild-type SA (Map⁺SA) compared to mice infected and reinfected withMap⁺SA or mice receiving a single inoculum of Map⁺SA. Evidence linkinginteractions between Map and T cells came from experiments in which nudemice were infected with Map⁻SA. The severity of osteomyelitis andarthritis was greater in nude mice compared to genotype controlsinfected with SA⁻Map, suggesting not only a role for T cells inprotection against SA infections but also a role for Map incircumventing T cell-mediated immunity. Testing the hypothesis that Mapacts to interfere with cellular immunity, various T cell-mediatedresponses were measured in vivo and in vitro in the presence of Map.DTH, which is a CD4⁺-mediated response, was significantly reduced inMap-treated mice and T cell proliferation in vitro was significantlyreduced in the presence of Map, likely as a function of Map-inducedapoptosis. These data evidence that Map is a virulence factor whoseabilities to potentially alter T cell function in vivo may affect SApersistence and survival and may function in facilitating recurring SAinfections.

[0047] Materials and Methods

[0048] Mice

[0049] Specific pathogen-free (MTV⁻) BALB/c and C3H/Hen mice werepurchased from Harlan Sprague Dawley, Indianapolis, Id. The animals weremaintained in facilities approved by the American Association forAccreditation of Laboratory Animal Care in accordance with currentregulations and standards of the United States Department ofAgriculture, Department of Health and Human Services, and NationalInstitutes of Health. All animal procedures were approved by theInstitutional Animal Care and Use Committee. Female mice were 8-10 weeksold at the start of each experiment.

[0050] Expression and Purification of Recombinant Proteins

[0051] Recombinant Map19, DbpA SdrF, M55, CNA, ACE19 and ACE40 wereexpressed in E. coli (JM101) (Qiagen, Chatsworth, Calif.) harboring theappropriate plasmid (11-16). E. coli was grown at 37° C. in LBcontaining the appropriate antibiotics until they reached an A₆₀₀ of 0.6(17). Isopropyl-β-D-thiogalactopyranoside (IPTG) (Life Technologies) wasadded to a final concentration of 0.2 mM, and the cells were incubatedat 37° C. for an additional 4 hours. Cells from a 1 L culture wereharvested by centrifugation and resuspended in 10 ml “binding buffer”(BB) (20 mM Tris HCl, 0.5 M NaCl, 15 mM imidazole, pH 8.0) and lysed ina French pressure cell at 11,000 pounds/inch² (13). The lysate wascentrifuged at 40,000×g for 15 min and the supernatant filtered througha 0.45 μm filter. A 1 ml iminodiacetic acid Sepharose column (Sigma, St.Louis, Mo.) was charged with 75 mM NiCl₂.6H₂O and equilibrated with BB.The filtered supernatant was applied to the column and washed with 10volumes of BB, then 10 volumes of BB containing 60 mM imidazole. Thebound proteins were eluted with BB containing 200 mM imidazole, dialyzedagainst PBS containing 10 mM EDTA, then dialyzed against PBS (13).Protein concentrations were determined by the Bicinchoninic Acid (BCA)Protein Assay (Pierce) and proteins were stored at −20° C. until use.

[0052] Quantitation of S. aureus and Intravenous Injections

[0053] Map⁺SA and Map⁻SA (strain Newman 8325) were grown overnight inLennox broth (LB) (Difco, Detroit, Mich.) media at 37° C. with shakingand used in all infection experiments. 50 μl of this culture was used toinoculate 10 ml of fresh LB in a 250 ml Erlenmeyer flask. The newcultures were grown as above until the optical density reached 0.5 at600 nm with a 1-cm quartz cuvette. Aliquots of each culture werequantified for colony forming units (CFU). The remainder of each culturewas washed three times in sterile PBS. The cultures, based on priorgrowth-curve determinations, were diluted to approximate 2×10⁷ CFU/ml.Mice were in injected i.v. with 1×10⁷ S. aureus in 0.5 ml PBS andmonitored for up to eight weeks. At the conclusion of the experiment,mice were sacrificed and the joints were examined histologically forarthritis development as described previously (18, 19).

[0054] Extraction of Map from Staphylococcus aureus

[0055] Map⁺SA and Map⁻SA were grown overnight as described above.Bacteria were pelleted by centrifugation and resuspended in 1 M LiCl(one tenth of the original media volume). The suspension was incubatedat 42° C. with shaking for two hours. The bacteria were pelleted and thesupernatant was removed and quantified for protein by UVspectrophotometry using 1 M LiCl as a blank. Extracted proteins werediluted to 0.2-mg/ml in PBS and passed through a 0.45-micron filter forsterilization prior to i.p. injection (7).

[0056] In Vitro Proliferation of BAT2.2 T Cells

[0057] The Borrelia burgdorferi-specific T cell line BAT2.2 wasstimulated with whole, inactive Borrelia and antigen presenting cells(APC) as described previously (18, 20). Briefly, 1×10⁵ BAT2.2 T cellswere cultured in 96-well flat-bottom plates (Costar, Cambridge Mass.)along with 3×10⁵ mitomycin-treated APC in complete medium (CTL) (RPMI1640 containing 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 50 μg/ml gentamicin, 0.2 mM nonessential amino acids, 11μg/ml sodium pyruvate, 0.02 MN-2-hydroxyethylpiperaxine-N′-2ethanesulfonic acid, and 5×10⁻5 N2-mercaptoethanol+10% heat-inactivated fetal bovine serum), and Borrelia(2 μg) in the presence of various proteins. Each treatment group wasdone in triplicate in a final volume of 200 μl complete medium. 10, 50,and 100 μg of each protein was added to each well and the T cells wereallowed to proliferate for 24-48 ours at 37° C. 4 h before the end ofthe proliferation period, 20 μl/well of3-{4,5-Dimethylthizol-2-y}-2,5diphenyl-tetraxolium bromide (MTT) (5mg/ml) was added to each well. After 4 h incubation at 37° C., 100 μl ofsolubilization buffer (0.04 N HCl in isopropanol) was added to each welland absorbance measured at 590 nm. Data are expressed as mean±SE of themean of triplicate wells.

[0058] Delayed Type Hypersensitivity (DTH) Assay

[0059] Mice were immunized with 20 μg of decorin binding protein A(DbpA) in complete Freund's adjuvant (day 0) (19). 7 days postimmunization, mice were challenged with 2.5 μg DbpA (13). DbpA wasadministered in 50 μl of PBS. At the time of immunization, days 2, 4,and 6 post immunization, mice were injected i.p. with 100 μg of nativeMap (N-Map) extracted from Map⁺SA, supernatant from Map⁻SA, or with 100μg of the recombinant proteins Map19, SdrF, M55 or ACE40 in 500 μl ofPBS (11-15, 21). The footpads were measured before challenge and 24 hlater, using a spring-loaded micrometer (Mitutoyo, Tokyo, Japan). Micewere anesthetized with Metofane™ during footpad measurements (22).

[0060] Adoptive T Cell Transfer

[0061] BALB/c mice (5 mice/group) were immunized with DbpA and weretreated with recombinant Map19 or recombinant ACE19 as described above.The day after the last Map19 or ACE40 treatment mice were sacrificed andthe spleens from each treatment group were enriched for T cells bypassage over nylon wool columns as described previously (20). 24 h afteri.p. injection of T cells (5×10⁷ nylon wool-enriched T cells/mouse in500 μl complete media), mice were challenged in the hind footpads withDbpA and the DTH response was assessed as described above.

[0062] Map-Induced Apoptosis of BAT2.2 Cells

[0063] 2×10⁶ BAT2.2 T cells/well (5 U IL-2/ml) were incubated in thepresence of Map19 or ACE19 in a final volume of 200 μl complete mediaand examined for apoptosis using an Apoptotic DNA Ladder Kit (RocheMolecular Biochemicals, Indianapolis, Ind.) according to manufacturersinstructions. 100 μg of each protein was used and apoptosis measuredafter a 24 h incubation at 37° C. DNA was treated with 2 μg/ml RNase(DNase free) for 20 min. at room temperature before examination byagarose gel electrophoresis.

[0064] Flow Cytometry

[0065] Nylon wool enriched T cells (1×10⁶/tube) were washed in PBScontaining 3% FBS and stained with the following monoclonal antibodies:fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8a (Ly2) andphycoerythrin (PE)-conjugated anti-mouse CD4 (L3T4) (PharMingen, SanDiego, Calif.). The cells were incubated with the directly conjugatedantibodies for 1 h at 4° C. and then washed and analyzed on a CoulterEpicProfile (Coulter Corp., Miami, Fla.).

[0066] Results

[0067] Experimental S. aureus infection

[0068] Infection parameters that resulted in high degrees of arthritisincidence were used to examine what role Map played in SA infection(23). BALB/c mice were injected in the tail i.v. with 1×10⁷ SA andsacrificed 4 weeks later for histological examination of hindtibiotarsal joints. These preliminary experiments revealed thatMap⁻SA-infected mice had both a reduced frequency and severity ofarthritis compared to Map⁺SA-infected controls. The hypothesis that Mapacted as an immunomodulator resulting in impaired immunity to SA with aconcomitant inability to respond to a challenge infection was tested byinfecting mice with Map⁻SA and Map⁺SA respectively, and challenging bothgroups with Map⁺SA 4 weeks later. Significant differences were observedin abscess formation in hearts and kidneys between the Map^(−/Map)⁺-infected group and the Map⁺/Map⁺- and -/Map⁺-infected groups (TableI). Less than 50% of hearts and 25% of kidneys from Map⁻/Map⁺ infectedmice presented with abscesses compared to >75% abscess formation in bothhearts and kidneys from Map⁺/Map⁺ and -/Map⁺ infected mice (Table I).Significant differences were also observed in arthritis andosteomyelitis scores and frequencies (Table II). Arthritis was prevalentin 54% of mice infected with Map⁻/Map⁺ compared to >80% incidence inMap⁺/Map⁺ and -/Map⁺ infected mice (Table II). The mean arthritis andosteomyelitis scores recorded were also more than 2 times less inMap⁻/Map⁺ infected mice compared to scores from Map⁺/Map⁺- and-/Map⁺-infected mice (Table II).

[0069] Map-mediated Inhibition of Delayed-type-hypersensitivity (DTH)

[0070] The similarity between Map and the peptide-binding region ofclass II MHC combined with the high levels of Map recoverable from thesurface of SA prompted experiments designed to address the questionregarding the potential role of Map on cellular immunity (7, 8). DTHresponses are initiated and mediated by CD4⁺ T cells in response torecall antigens and result in specific, measurable inflammation at thesite of challenge. Mice immunized with recombinant decorin-bindingprotein A (DbpA) emulsified in complete Freund's adjuvant (CFA)developed a significant DTH response to DbpA as measured by footpadswelling 7 days post immunization (FIG. 1) (19). However, mice treatedwith native Map (⁺Map Supernatant) or recombinant Map19 on the day ofimmunization (day 0) and days 2, 4 and 6 post immunization had asignificantly reduced DTH response to DbpA compared to control mice(FIG. 1). Neither supernatants from Map⁻SA (FIG. 1a) or recombinantcontrol protein ACE19 had any measurable effects on the DTH response toDbpA (FIGS. 1b-c). Map19's inhibitory effects were not affected bygenetic differences since the DTH response was diminished in both BALB/cand C3H/Hen mice following immunization and challenge (FIGS. 1b and c,respectively).

[0071] Map Time Course and Dose Response for DTH Inhibition

[0072] Both the induction and elicitation of the DTH response wereaffected by Map treatment since Map19 injected either before or afterimmunization resulted in a significant reduction in the DTH response(Table III). Although all Map19-treated mice had a significantly reducedresponse to DbpA challenge following immunization, mice receiving Map19on both the day of immunization and challenge (in addition to d2 and d4,Experiment I Table III) had the greatest reduction in footpad swellingcompared to control mice (13.7±1.46 vs. 34.75±3.47 mm×10⁻²,respectively) (Experiment I, Table III). The hypothesis that Map19 couldact to prevent DTH by interfering with either the induction orelicitation of DTH was tested by comparing challenge responses inuntreated mice to groups either treated with Map19 every other day(starting on the day of immunization) or to mice treated with Map19 onlyon the day of immunization and challenge (Experiment 2, Table III).Map19-treated mice had a significantly reduced DTH response compared tountreated or ACE40-treated controls (Experiment II, Table III). Sincemice treated only on the days of immunization and challenge had asignificantly reduced DTH response indistinguishable from the responseobserved in mice treated with Map19 every other day, it evidenced thatMap19's inhibitory activity correlated with T cell activation and thatit's capacity to interfere with T cell function was maximal during the Tcell activation stages of DTH. Doses of Map in the excess of 100 μg didnot further reduce the DTH response, however, 25 μg, the lowest dosedtested in this experiment, still significantly reduced the DTH response(FIG. 2).

[0073] Adoptively Transferred T Cells from Map-treated Mice

[0074] Mice immunized with DbpA were either left untreated or injectedi.p. with either Map19 or the recombinant control protein SdrF on theday of immunization (day 0) and on days 2, 4, and 6 post immunization.On day 7, mice were sacrificed and single cell suspensions from wholespleens were prepared and enriched for T cells by passage over nylonwool columns (20). Adoptive transfer of nylon wool-purified T cells fromMap19-treated mice did not elicit a DTH response to DbpA in naiverecipients compared to mice adoptively transferred with enriched T cellsfrom control groups (FIG. 3). Flow cytometric analysis of cells nylonwool-collected cells revealed a profile that was 46.83±0.92% CD4⁺,31.63±0.96% CD8⁺, 1.2±0.26% CD4⁺ CD8⁺, and 20.4±1.33% CD4⁻ CD8⁻. Thesedata are expressed as the mean percentage of positive cells±SE for the 3groups examined.

[0075] Inhibition of T Cell Proliferation and Apoptosis Induction inVitro.

[0076] Recombinant Map10 (SEQ ID NOS. 3 and 4) and Map19 (SEQ ID NOS. 1and 2) were tested for their ability to inhibit the proliferation of theBorrelia-specific T cell line BAT 2.2 (8, 20). T cell proliferation wasmeasured at 40 h after plating in the presence of mitomycin C-treatedsyngeneic antigen presenting cells (APC) and inactive Borrelia (iBb)(20). Proliferation was measured as a function of tetrazolium blueproduction following a 4 h incubation in the presence of MTT. BAT 2.2cells in the presence of either Map10 or 19 but not in the presence ofrecombinant control proteins CNA or M55 were inhibited fromproliferating (FIG. 4) (24). BAT 2.2 incubated in the presence ofBorrelia only were plotted as baseline as the control group with thehighest background proliferation (FIG. 4). In a similar experiment,BAT2.2 cells in the presence of IL-2 were cultured in the presence ofMap19 for 24 h. DNA extracted from BAT2.2 T cells incubated in thepresence of Map19 was examined for fragmentation by gel electrophoresis(FIG. 5). DNA fragmentation comparable to apoptotic-positive control DNA(lane 5) was only observed in DNA extracted from Map19-treated T cells(lane 3) but not untreated (lane 2) or ACE40-treated (lane 4) T cells(FIG. 5).

[0077] Summary

[0078] Reinfection of humans with SA is one of the hallmarks of diseasescaused by this pathogen and the roles of acquired and innate immunity inprotection against infection vary with the many manifestation of diseaseresulting from SA infections (25-28). While SA infections affecting theskin appear to be exacerbated by strong cellular responses, it is clearthat cellular immunity is critical in orchestrating the clearance ofsystemic SA infections and in preventing reinfection with the same orsimilar pathogens (29-33). One possible reason for recurring SAinfections is the reduction in chemotactic, phagocytic and bactericidalfunctions of polymorphonuclear leukocytes from patients with chronic orrecurrent SA infections (27, 30, 33). Whether this is a function of thebacterial infection or a preexisting condition in these individuals isnot known (27, 30, 33). Regardless, the presence of SA-immunoregulatorymolecules suggests that these bacteria have the potential to counteractor evade host defense mechanisms. Both superantigens and protein Aproduced by SA during an infection serve immune-evasion functions.Superantigens can activate between 5-20% of T cells by directly bindingto both the major histocompatibility complex (MHC) class II molecules onantigen-presenting cells and to the T cell receptor (TCR) on T cells.This interaction can initiate apoptosis in T cells and thymocytes invivo and in vitro. The in vivo effects of such massive T cellstimulation often results in disease (i.e. toxic shock syndrome and foodpoising in humans) (34). Protein A, while less harmful to the hostcompared to superantigens, may also serve as a means of immune evasionby binding to the Fc fragment of immunoglobulins (i.e. IgG) resulting inloss of antibody function.

[0079] The present series of tests supported the idea that Map mayfunction as an immune modulator with the capacity to affect host immuneresponses during SA infections. In addition to its potential role as abacterial adhesin; our tests showed that Map apparently has the capacityto interfere with T cell activation and/or proliferation facilitating SAsurvival in mammals (8, 11, 24, 35, 36). Sequence analysis of the SAgenome revealed 5 open-reading frames encoding Map-like proteins (14).While only one of these Map proteins (SA1751) had a >80% homology toNewman stain Map (8, 14), the presence of other Map-like proteinssuggested a critical role for Map in SA survival; perhaps the potentialto encode a variety of MHC II-like proteins can serve to potentiatesurvival in mammals of varied genetic backgrounds.

[0080] Additional evidence suggesting Map serves as an immunomodulatoryprotein stemmed from double infection studies in which a primaryinfection with Map⁻SA conferred significant protection againstreinfection with Map⁺SA. This contrasts significantly with SA-inducedpathology from mice receiving primary and secondary infections withMap⁺SA. Accordingly, it appears that T cell-mediated responses inMap⁺SA-infected mice are abrogated by the presence of Map compared toMap⁻SA-infected mice which develop cell-mediated immunity over thecourse of infection. Map⁻SA infection, which is cleared over time,results in a memory response capable of controlling a secondary Map⁺SAinfection. That a primary Map⁻SA infection conferred significant but notcomplete protection against Map⁺SA challenge suggested that the delicatebalance between an anamnestic response and Map-mediated immunomodulationcould be affected by the challenge dose. Our tests showed inhibition ofDTH responses directly or as a result of adoptively transferred T cellsfrom Map-treated mice, and this combined with the in vitro effects ofMap on T cell proliferation evidenced a direct involvement of Map with Tcells resulting in apoptosis. Flow cytometric analysis of fluoresceinisothiocyanate (FITC)-labeled Map19 revealed binding to 100% of BAT2.2 Tcells (data not shown).

[0081] In additional tests evidencing the effect on the Map protein on Tcell-mediated responses, nylon wool-purified naive T cells cultured inthe presence of Map19 were not induced to either proliferate or undergoapoptosis. Furthermore, proliferation of naive T cells as a result ofincubation with concanavalin A or by antibody-cross-linking of the TCRwas not inhibited by Map. This evidence that activated T cells but notnaive T cells are susceptible to Map and that T cell proliferation via‘non classical’ pathways bypasses the Map-mediated inhibition of T cellproliferation. Based on Map's effects on cellular immune responses invivo and in vitro, it appears that this protein is yet another weaponused by SA to escape immune recognition and clearance. Accordingly, inaccordance with the present invention, the administration of effectiveamounts of the Map protein or its active regions or fragments such asMap19 appears to be useful in achieving the suppression or modulation ofT cell-mediated responses to a host cell against S. aureus and thus maybe useful in methods to prevent or reduce the persistence or virulenceof infection by staphylococcal bacteria.

Example 2 Tests of Map, Map10 and Map19

[0082] Materials and Methods

[0083] Mice

[0084] Specific pathogen-free (MTV⁻) BALB/c were purchased from HarlanSprague Dawley, Indianapolis, Ind. The animals were maintained infacilities approved by the American Association for Accreditation ofLaboratory Animal Care in accordance with current regulations andstandards of the United States Department of Agriculture, Department ofHealth and Human Services, and National Institutes of Health. All animalprocedures were approved by the Institutional Animal Care and UseCommittee. Female mice were 8-10 weeks old at the start of eachexperiment.

[0085] Quantitation of S. aureus and Intravenous Injections

[0086] Map⁺SA and Map⁻ SA (strain Newman or 8325) were grown overnightin Lennox broth (LB) (Difco, Detroit, Mich.) media at 37° C. withshaking. 50 μl of this culture was used to inoculate 10 ml of fresh LBin a 250 ml Erlenmeyer flask. The new cultures were grown as above untilthe optical density reached 0.5 at 600 nm with a 1-cm quartz cuvette.Aliquots of each culture were quantified for colony forming units (CFU).The remainder of each culture was washed three times in sterile PBS. Thecultures, based on prior growth-curve determinations, were diluted toapproximate 2×10⁷ or 2×10⁶ CFU/ml. CFU was determined for each of thediluted cultures. Mice were in injected i.v. with either 5×10⁶ or 1×10⁷S. aureus in 0.5 ml PBS and monitored for 4 weeks. At the conclusion ofthe experiment mice were sacrificed and the joints were examinedhistologically for arthritis development as described previously (14).

[0087] Extraction of Map from Staphylococcus aureus

[0088] Map⁺ SA and Map⁻ SA strain Newman were grown overnight withshaking in LB media. Bacteria were pelleted by centrifugation andresuspended in 1 M NaCl (one tenth of the original media volume). Thesuspension was incubated at 42° C. with shaking for two hours. Thebacteria were pelleted and the supernatant was removed and quantifiedfor protein by UV spectrophotometry using 1 M NaCl as a blank. Extractedproteins were diluted to 0.2-mg/ml in PBS and passed through a0.45-micron filter for sterilization prior to i.p. injections. FinalNaCl concentrations of the diluted extracts approximated twice that ofphysiologic conditions (320 mM compared to 150 mM) since the originalextracts were usually diluted 1:5.

[0089] In Vitro Proliferation of BAT2.2 T Cells

[0090] The Borrelia burgdorferi-specific T cell line BAT2.2 wasstimulated with whole, inactive Borrelia and antigen presenting cells(APC) as described previously (14). Briefly, 1×10⁵ BAT2.2 T cells werecultured in 96-well flat-bottom plates (Costar, Cambridgem Mass.) alongwith 3×10⁵ mitomycin-treated APC in complete medium (RPMI 1640containing 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 50 μg/ml gentamicin, 0.2 mM nonessential amino acids, 11μg/ml sodium pyruvate, 0.02 MN-2-hydroxyethylpiperaxine-N′-2ethanesulfonic acid, and 5×10⁻5 N2-mercaptoethanol+10% heat-inactivated fetal bovine serum), and Borrelia(2 μg) in the presence of various proteins. Each treatment group wasdone in triplicate in a final volume of 200 μl complete medium. 10, 50,and 100 μg of each protein was added to each well and the T cells wereallowed to proliferate for 40-72 hours at 37° C. 4 h before the end ofthe proliferation period, 20 μl/well of3-{4,5-Dimethylthizol-2-y}-2,5diphenyl-tetraxolium bromide (MTT) (5mg/ml) was added to each well. After 4 h incubation at 37° C., 100 μl ofsolubilization buffer (0.04 N HCl in isopropanol) was added to each welland absorbance measured at 570 nm. Data are expressed as mean±SE of themean of triplicate wells.

[0091] Delayed Type Hypersensitivity (DTH) Assay

[0092] Mice were immunized with 20 μg of decorin binding protein A(DbpA) in complete Freund's adjuvant. 7 days post immunization, micewere challenged with 2.5 μg DbpA. DbpA was administered in 50 μl of PBS.At the time of immunization, days 3, 5, and 7 post immunization, micewere injected i.p. in 500 μl of PBS with 100 μg of native Map (N-Map)extracted from Map⁺ SA, supernatant from Map⁻SA, or the recombinantproteins Map 10 or SdrF. The footpads were measured before challenge and24 h later, using a spring-loaded micrometer (Mitutoyo, Tokyo, Japan).Mice were anesthetized with Metofane™ during footpad measurements.

[0093] Expression and Purification of Recombinant Proteins

[0094] Recombinant Map10, Map19, CAN, SdrF, and M55 were expressed in E.coli (JM101) (Qiagen, Chatsworth, Calif.) harboring the appropriateplasmid. E. coli was grown at 37 C in LB containing the appropriateantibiotics until they reached an A₆₀₀ of 0.6 (15).Isopropyl-D-thiogalactopyranoside (IPTG) (Life Technologies) was addedto a final concentration of 0.2 mM, and the cells were incubated at 37°C. for an additional 4 hours. Cells from a 1 L culture were harvested bycentrifugation and resuspended in 10 ml “binding buffer” (BB) (20 mMTris HCl, 0.5 M NaCl, 15 mM imidazole, pH 8.0) and lysed in a Frenchpressure cell at 11,000 pounds/inch². The lysate was centrifuged at40,000×g for 15 min and the supernatant filtered through a 0.45 μmfilter. A 1 ml iminodiacetic acid Sepharose column (Sigma, St. Louis,Mo.) was charged with 75 mM NiCl₂.6H₂O and equilibrated with BB. Thefiltered supernatant was applied to the column and washed with 10volumes of BB, then 10 volumes of BB containing 60 mM imidazole. Thebound proteins were eluted with BB containing 200 mM imidazole, dialyzedagainst PBS containing 10 mM EDTA, then dialyzed against PBS. Theprotein concentration was determined by the Bicinchoninic Acid (BCA)Protein Assay (Pierce, Rockford, Ill.) and proteins were stored at −20°C.

[0095] Map-induced Apoptosis of BAT2.2 Cells

[0096] 2×10⁶ BAT2.2 T cells/well (5 U IL-2/ml) were incubated in thepresence of either anti-T cell receptor chain antibody (5 or 10 μg/well)(clone H57-597, Pharmingen, San Diego, Calif.), Map10, Map19, or M55 ina final volume of 200 μl complete media and examined for apoptosis usingan Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals, Indianapolis,Ind.) according to manufacturers instructions. 100 μg of each proteinwas used and apoptosis measured after a 24 h incubation at 37° C. DNAwas treated with 2 μg/ml RNase (DNase free) for 20 min. at roomtemperature before examination by agarose gel electrophoresis.

[0097] Adoptive T Cell Transfer

[0098] BALB/c mice (5 mice/group) were immunized with DbpA and weretreated with recombinant Map10 or recombinant SdrF as described above.The day after the last Map10 or SdrF treatment (day 8 post immunization)mice were sacrificed and the spleens from each treatment group wereenriched for T cells by passage over nylon wool columns as describedpreviously (14). 24 h after i.p. injection of T cells (5×10⁷ nylonwool-enriched T cells/mouse in 500 μl complete media), mice werechallenged in the hind footpads with DbpA and the DTH response wasassessed as described above.

[0099] Flow Cytometry

[0100] Nylon wool enriched T cells (1×10⁶/tube) were washed in PBScontaining 3% FBS and stained with the following monoclonal antibodies:fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8a (Ly2) andphycoerythrin (PE)-conjugated anti-mouse CD4(L3T4) (PharMingen, SanDiego, Calif.). The cells were incubated with the directly conjugatedantibodies for 1 h at 4° C. and then washed and analyzed on a CoulterEpicProfile (Coulter Corp., Miami, Fla.).

[0101] Results

[0102] Experimental S. aureus Infection

[0103] Map⁺ or Map⁻ strains of SA Newman or 8325 were administered i.v.to BALB/c mice and monitored for four weeks. Mortality between groupsover a 4 week period was similar, however, arthritis development wassignificantly different between groups. Mice infected with Map⁻ SA had amean arthritis score of 0.5 and an arthritis incidence of 50%, comparedto an arthritis score of 2.35 and 2.25 for Map⁺ strains Newman and 8325,respectively. The incidence of arthritis was also 75% in mice infectedwith Map⁺ SA, however only Map⁺ Newman strain-infected mice developedsever osteomyelitis (75%) compared to mice from other groups (0%).Furthermore, spleens harvested from strain Newman Map⁺ SA-infected micewere significantly larger than spleens isolated from mice infected withstrain Newman Map⁻ SA four weeks post infection.

[0104] Inhibition of T Cell Proliferation in Vitro.

[0105] Recombinant Map 10 and Map 19 were tested for their ability toinhibit the proliferation of the Borrelia-specific T cell line BAT 2.2(13, 14). T cell proliferation was measured at 40 and 49 h after platingin the presence of mitomycin C-treated syngeneic antigen presentingcells (APC) and inactive Borrelia (iBb) (14). Proliferation was measuredas a function of tetrazolium blue production following a 4 h incubationin the presence of MTT. BAT 2.2 cells in the presence of either Map10 or19 but not in the presence of recombinant control proteins CNA or M55were inhibited from proliferating (3). BAT 2.2 incubated in the presenceof Borrelia only were plotted as baseline since this control group hadthe highest background proliferation.

[0106] Inhibition of T Cell Activity in Vivo.

[0107] N-Map and recombinant Map 10 were tested for their ability tointerfere with the elicitation of a DTH response to DbpA inDbpA-immunized mice. On the day of immunization and on days 3, 5, and 7post immunization mice were injected i.p. with 100 μg (500 μl) of eitherN-Map, Map supernatant, Map 10, or SdrF. At day 7post immunization, micefrom all groups were challenged in the hind footpads with 2.5 μg iBb.Footpads were measured 0 and 24 h post challenge. Mice treated witheither N-Map or Map 10 had a significantly reduced DTH response to DbpAcompared to untreated, Map⁻ supernatant or SdrF-treated mice.

[0108] Adoptively Transferred T Cells from Map-treated Mice do notElicit a DTH

[0109] Mice immunized with DbpA were either left untreated or injectedi.p. with either Map 10 or the recombinant control protein SdrF on theday of immunization and on days 3, 5, and 7 post immunization. On day 8,mice were sacrificed and single cell suspensions from whole spleens wereprepared and enriched for T cells by passage over nylon wool columns(14). Adoptive transfer of nylon wool purified T cells from Map10-treated mice did not elicit a DTH response to DbpA in naïverecipients compared to mice adoptively transferred with enriched T cellsfrom control groups. Flow cytometric analysis of cells nylonwool-collected cells revealed a profile that was 46.83±0.92% CD4⁺,31.63±0.96% CD8⁺, 1.2±0.26% CD4⁺ CD8⁺, and 20.4±1.33% CD4⁻CD8⁻. Thesedata are expressed as the mean percentage of positive cells±SE for the 3groups examined. TABLE I Abscess formation in heart and kidneysharvested from Map⁻ and Map⁺ SA-infected mice^(A) Tissue Examined^(B)Infecting Strains Heart Kidneys Map−/Map⁺ 12/26 (46%)^(C,D) 13/52(25%)^(E) Map⁺/Map⁺ 17/19 (89%) 33/38 (86%) −/Map⁺ 29/31 (94%) 48/62(77%)

[0110] TABLE II Histological examination of joints harvested from Map⁻and Map⁺ SA-infected mice^(A) Mean Arthritis Arthritis Mean OstomylitisOsteomyelitis Infecting Strains Rating Frequency (%) Score Frequency (%)Map−/Map+ 0.84^(B) 14/26 (54%)^(C) 0.57^(B)  6/26^(D) (23%) Map+/Map+1.65 18/21 (86%) 1.95 14/21 (66%) −/Map+ 2.06 28/32 (88%) 1.48 14/32(44%)

[0111] TABLE III Histological examination of joints harvested from SA⁻Map or SA⁺ Map-infected nude mice^(A) Mean Arthritis Arthritis MeanOstomylitis Osteomyelitis Infecting Strains Rating Frequency (%) ScoreFrequency (%) nu/+/Map⁺SA 2.86 7/7 (100%) 2.29 5/7 (71%) nu/+/Map⁻SA1.33 8/9 (89%)^(C) 0.44^(B) 3/9 (33%) nu/nu/Map⁺SA 2.43 8/8 (100%) 2.628/8 (100%) nu/nu/Map⁻SA 2.10 7/10 (70%) 1.20 6/10 (60%)

[0112] TABLE IV The Effect of Map19 Treatment at Various Times Beforeand After Immunization on the Elicitation of DTH^(A) Mean Treatment^(B)Time Course Footpad Exp. I d-6 d-4 d-2 d0^(E) d2 d4 d7^(F) Swelling^(C)SE^(D) Map19 + + + +IM CH 18.75^(G) ±3.26 Map19 + + +IM CH 22.75^(G)±2.66 Map19 + +IM CH 20.25^(G) ±1.93 Map19 +IM CH 23.00^(G) ±1.36 Map19+IM + CH 17.75^(H) ±2.06 Map19 +IM + + CH 23.62^(G) ±3.48 Map19 +IM + ++CH 13.75^(I) ±1.46 — CH  5.50^(I) ±1.24 — IM CH 34.75 ±3.47 Exp. IIMap19 +IM + + +CH 13.30^(I) ±1.50 Map19 +IM +CH 10.10^(I) ±0.82 ACE40+IM + + +CH 26.60^(J) ±2.83 ACE40 +IM +CH 26.75^(J) ±1.73 — CH  3.10^(I)±0.67 — IM CH 33.56 ±3.04

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1 4 1 603 DNA Staphylococcus aureus CDS (1)..(603) 1 atg aga gga tcg catcac cat cac cat cac gga tcc cag att cca tat 48 Met Arg Gly Ser His HisHis His His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 aca atc act gtg aatggt aca agc caa aac att tta tca agc tta aca 96 Thr Ile Thr Val Asn GlyThr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30 ttt aat aag aat caa caaatt agt tat aaa gat ata gag aat aaa gtt 144 Phe Asn Lys Asn Gln Gln IleSer Tyr Lys Asp Ile Glu Asn Lys Val 35 40 45 aaa tca gtt tta tac ttt aataga ggt att agt gat atc gat tta aga 192 Lys Ser Val Leu Tyr Phe Asn ArgGly Ile Ser Asp Ile Asp Leu Arg 50 55 60 ctt tct aag caa gca aaa tac acggtt cat ttt aag aat gga aca aaa 240 Leu Ser Lys Gln Ala Lys Tyr Thr ValHis Phe Lys Asn Gly Thr Lys 65 70 75 80 aga gtt gtc gat ttg aaa gca ggcatt cac aca gcc gac tta atc aat 288 Arg Val Val Asp Leu Lys Ala Gly IleHis Thr Ala Asp Leu Ile Asn 85 90 95 aca agt gac att aaa gca att agt gttaac gta gat act aaa aag caa 336 Thr Ser Asp Ile Lys Ala Ile Ser Val AsnVal Asp Thr Lys Lys Gln 100 105 110 gtg aaa gat aaa gag gca aaa gca aatgtt caa gtg ccg tat aca atc 384 Val Lys Asp Lys Glu Ala Lys Ala Asn ValGln Val Pro Tyr Thr Ile 115 120 125 act gtg aat ggt aca agc caa aac atttta tca aac tta aca ttt aaa 432 Thr Val Asn Gly Thr Ser Gln Asn Ile LeuSer Asn Leu Thr Phe Lys 130 135 140 aag aat cag caa att agt tat aaa gattta gag aat aat gta aaa tca 480 Lys Asn Gln Gln Ile Ser Tyr Lys Asp LeuGlu Asn Asn Val Lys Ser 145 150 155 160 gtt tta aaa tca aac aga ggt ataact gat gta gat tta aga ctt tca 528 Val Leu Lys Ser Asn Arg Gly Ile ThrAsp Val Asp Leu Arg Leu Ser 165 170 175 aaa caa gcg aaa ttt aca gtt aatttt aaa aat ggc acg aaa aaa gtt 576 Lys Gln Ala Lys Phe Thr Val Asn PheLys Asn Gly Thr Lys Lys Val 180 185 190 atc gat ttg aaa gca ggc att tattga 603 Ile Asp Leu Lys Ala Gly Ile Tyr 195 200 2 200 PRT Staphylococcusaureus 2 Met Arg Gly Ser His His His His His His Gly Ser Gln Ile Pro Tyr1 5 10 15 Thr Ile Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser Ser LeuThr 20 25 30 Phe Asn Lys Asn Gln Gln Ile Ser Tyr Lys Asp Ile Glu Asn LysVal 35 40 45 Lys Ser Val Leu Tyr Phe Asn Arg Gly Ile Ser Asp Ile Asp LeuArg 50 55 60 Leu Ser Lys Gln Ala Lys Tyr Thr Val His Phe Lys Asn Gly ThrLys 65 70 75 80 Arg Val Val Asp Leu Lys Ala Gly Ile His Thr Ala Asp LeuIle Asn 85 90 95 Thr Ser Asp Ile Lys Ala Ile Ser Val Asn Val Asp Thr LysLys Gln 100 105 110 Val Lys Asp Lys Glu Ala Lys Ala Asn Val Gln Val ProTyr Thr Ile 115 120 125 Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser AsnLeu Thr Phe Lys 130 135 140 Lys Asn Gln Gln Ile Ser Tyr Lys Asp Leu GluAsn Asn Val Lys Ser 145 150 155 160 Val Leu Lys Ser Asn Arg Gly Ile ThrAsp Val Asp Leu Arg Leu Ser 165 170 175 Lys Gln Ala Lys Phe Thr Val AsnPhe Lys Asn Gly Thr Lys Lys Val 180 185 190 Ile Asp Leu Lys Ala Gly IleTyr 195 200 3 396 DNA Staphylococcus aureus CDS (1)..(396) 3 atg aga ggatcg cat cac cat cac cat cac gga tcc cag att cca tat 48 Met Arg Gly SerHis His His His His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 aca atc actgtg aat ggt aca agc caa aac att tta tca agc tta aca 96 Thr Ile Thr ValAsn Gly Thr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30 ttt aat aag aatcaa caa att agt tat aaa gat ata gag aat aaa gtt 144 Phe Asn Lys Asn GlnGln Ile Ser Tyr Lys Asp Ile Glu Asn Lys Val 35 40 45 aaa tca gtt tta tacttt aat aga ggt att agt gat atc gat tta aga 192 Lys Ser Val Leu Tyr PheAsn Arg Gly Ile Ser Asp Ile Asp Leu Arg 50 55 60 ctt tct aag caa gca aaatac acg gtt cat ttt aag aat gga aca aaa 240 Leu Ser Lys Gln Ala Lys TyrThr Val His Phe Lys Asn Gly Thr Lys 65 70 75 80 aga gtt gtc gat ttg aaagca ggc att cac aca gcc gac tta atc aat 288 Arg Val Val Asp Leu Lys AlaGly Ile His Thr Ala Asp Leu Ile Asn 85 90 95 aca agt gac att aaa gca attagt gtt aac gta gat act aaa aag caa 336 Thr Ser Asp Ile Lys Ala Ile SerVal Asn Val Asp Thr Lys Lys Gln 100 105 110 gtg aaa gat aaa gag gca aaagca aat gtt gtc gac ctg cag cca agc 384 Val Lys Asp Lys Glu Ala Lys AlaAsn Val Val Asp Leu Gln Pro Ser 115 120 125 tta att agc tga 396 Leu IleSer 130 4 131 PRT Staphylococcus aureus 4 Met Arg Gly Ser His His HisHis His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 Thr Ile Thr Val Asn GlyThr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30 Phe Asn Lys Asn Gln GlnIle Ser Tyr Lys Asp Ile Glu Asn Lys Val 35 40 45 Lys Ser Val Leu Tyr PheAsn Arg Gly Ile Ser Asp Ile Asp Leu Arg 50 55 60 Leu Ser Lys Gln Ala LysTyr Thr Val His Phe Lys Asn Gly Thr Lys 65 70 75 80 Arg Val Val Asp LeuLys Ala Gly Ile His Thr Ala Asp Leu Ile Asn 85 90 95 Thr Ser Asp Ile LysAla Ile Ser Val Asn Val Asp Thr Lys Lys Gln 100 105 110 Val Lys Asp LysGlu Ala Lys Ala Asn Val Val Asp Leu Gln Pro Ser 115 120 125 Leu Ile Ser130

What is claimed is:
 1. A method of preventing or modulating a Tcell-mediated response in a host comprising administering to the host anisolated S. aureus Map protein in an amount effective to prevent ormodulate a T cell-mediated response in the host.
 2. An method accordingto claim 1 wherein the T cell-mediated response is DTH.
 3. A method oftreating or preventing pathogenic conditions associated withoverstimulation of T cells in a human or animal patient comprisingadministering to the host an isolated S. aureus Map protein in an amounteffective to treat or prevent a condition associated withoverstimulation of T cells.
 4. A method according to claim 3 wherein thecondition associated with overstimulation of T cells is selected fromthe group consisting of toxic shock syndrome and poison ivy.
 5. Apharmaceutical composition for preventing or modulating a Tcell-mediated response to a staphylococcal infection comprising anisolated S. aureus Map protein in an amount effective to prevent ormodulate a T cell-mediated response and a pharmaceutically acceptablevehicle, carrier or excipient.
 6. An isolated S. aureus Map19 protein.7. An isolated protein according to claim 6 having an amino acidsequence according to SEQ ID NO:
 2. 8. An isolated protein according toclaim 6 having an amino acid sequence encoded by a nucleic acid sequenceaccording to SEQ ID NO: 1 or degenerates thereof.
 9. A method ofpreventing or modulating a T cell-mediated response in a host comprisingadministering to the host an isolated S. aureus Map19 protein accordingto claim 6 in an amount effective to prevent or modulate a Tcell-mediated response in the host.
 10. A pharmaceutical composition forpreventing or modulating a T cell-mediated response to a staphylococcalinfection comprising an isolated S. aureus Map19 protein according toclaim 6 in an amount effective to prevent or modulate a T cell-mediatedresponse and a pharmaceutically acceptable vehicle, carrier orexcipient.
 11. A method of treating or preventing pathogenic conditionsassociated with overstimulation of T cells in a human or animal patientcomprising administering to the host an isolated S. aureus Map19 proteinaccording to claim 6 in an amount effective to treat or prevent acondition associated with overstimulation of T cells.
 12. A methodaccording to claim 11 wherein the pathogenic condition associated withoverstimulation of T cells is selected from the group consisting oftoxic shock syndrome and poison ivy.
 13. A pharmaceutical compositionfor preventing or modulating a T cell-mediated response to astaphylococcal infection comprising an isolated S. aureus Map10 proteinin an amount effective to prevent or modulate a T cell-mediated responseand a pharmaceutically acceptable vehicle, carrier or excipient.
 14. Amethod of preventing or modulating a T cell-mediated response in a hostcomprising administering to the host the composition of claim 13 in anamount effective to prevent or modulate a T cell-mediated response inthe host.
 15. A method of treating or preventing pathogenic conditionsassociated with overstimulation of T cells in a human or animal patientcomprising administering to the host an isolated S. aureus Map10 proteinin an amount effective to treat or prevent a condition associated withoverstimulation of T cells.
 16. A method of treating or preventing a Tcell lymphoproliferative disease comprising administering to the host anisolated Map protein selected from the the group consisting of the Mapprotein, Map10 protein and Map19 protein, in an amount effective totreat or prevent a T cell lymphoproliferative disease.